Effect of an NGR Peptide on the Efficacy of the Doxorubicin Phospholipid Delivery System.

This study is a continuation of an investigation into the effect of a targeted component, a peptide with an NGR, on the properties of the previously developed doxorubicin phospholipid delivery system. The NGR peptide has an affinity for aminopeptidase N (known as the CD13 marker on the membrane surface of tumor cells) and has been extensively used to target drug delivery systems. This article presents the results of a study investigating the physical properties of the phospholipid composition with and without the peptide chain: particle size, zeta potential, stability in fluids, and dependence of doxorubicin release from nanoparticles at different pH levels (5.0, 6.5, 7.4). The cytotoxic effect of the compositions has also been shown to depend on the dose of the drug used for incubation, the presence of the targeted component in the composition, and the time of incubation time of the substances. There was a significant difference in the cytotoxic effect on HT-1080 (CD13-positive) and MCF-7 (CD13-negative) cells. Cell death pathway analysis has shown that death occurred mainly by apoptosis. We also present data on the effect of doxorubicin embedded in phospholipid nanoparticles with the targeted peptide on DNA assessed by differential pulse voltammetry, the mechanism of action being electrostatic interactions. The interactions of native dsDNA with doxorubicin encapsulated in phospholipid nanoparticles with the targeted peptide were studied electrochemically by differential pulse voltammetry. Here, we have highlighted that the targeted peptide in the doxorubicin composition moved specific interaction of the drug with dsDNA from intercalative mode to electrostatic interactions.

Drug Transport System Based on Phospholipid Nanoparticles: Production Technology and Characteristics.

One of the current trends in modern pharmaceuticals is the supply of drugs by transport systems. The use of delivery systems allows to increase the therapeutic efficacy, tolerability, and safety of drug therapy. Liposomes, polymer nanoparticles, carbon nanoparticles, blood cells, metal nanoparticles, oxides, etc., are used as transport systems. This work is aimed at obtaining a finished technological product based on soy phospholipids with particle size in the nanometer range and reproducible characteristics (size, charge). For this purpose, we carried out investigations to select the optimal conditions of technological process. The developed technology makes it possible to obtain phospholipid nanoparticles without the use of any solubilizers and/or surfactants, which increases its practical relevance for further work. The versatility of the technology is demonstrated by the example of incorporation of drugs of various chemical nature and pharmacotherapeutic groups.

Chlorin e6 Phospholipid Delivery System Featuring APN/CD13 Targeting Peptides: Cell Death Pathways, Cell Localization, In Vivo Biodistribution.

We have previously designed a phospholipid delivery system for chlorin e6 to increase the efficacy of photodynamic therapy involving a second-generation photosensitizer. Further research into the matter led to double modification of the obtained nanoparticles with ligands exhibiting targeting and cell-penetrating effects: an NGR-containing peptide and heptaarginine (R7), respectively. This study investigated the cell death pathway on HT-1080 tumor cells after treatment with the proposed compositions: the chlorin e6 phospholipid composition and the two-peptide chlorin e6 phospholipid composition. It was demonstrated that most of the cells died by apoptosis. Colocalization analysis of chlorin e6 in the phospholipid composition with two peptides showed mitochondria are one of the targets of the photosensitizer. An HT-1080 tumor-bearing mouse model was used to evaluate the biodistribution of the drug in tumor, liver, and kidney tissues after administration of the study compositions in comparison with free chlorin e6. The photosensitizer mostly accumulated in the tumor tissue of mice administered the phospholipid compositions, and accumulation was increased 2-fold with the peptide-containing composition and approximately 1.5-fold with the unenhanced composition, as compared with free chlorin e6. The enhancement of the chlorin e6 phospholipid composition with targeting and cell-penetrating peptides was found to be effective both in vitro and in vivo.

Study of Physico-Chemical Properties and Morphology of Phospholipid Composition of Indomethacin.

Nonsteroidal anti-inflammatory drugs (NSAIDs), inhibitors of cyclooxygenase-2, an enzyme involved in the formation of anti-inflammatory prostaglandin PGE2, are the most common treatment for chronic inflammatory diseases, such as, for example, arthritis. One of the most commonly used drugs of this class is indomethacin, a derivative of indolylacetic acid. In this work, we studied the physicochemical properties of the phospholipid composition of indomethacin obtained earlier (codenamed "Indolip") and the effect of freeze drying on its parameters. It was shown that the properties such as particle size, light transmission, phospholipid oxidation index did not change significantly, which indicated the stability of the drug after lyophilization. Measurement of the spectra of small-angle neutron scattering has shown that morphologically, Indolip is a vesicle whose radius is five times greater than the value of the bilayer thickness.

Electrochemical studies of the interaction of rifampicin and nanosome/rifampicin with dsDNA.

The interactions of dsDNA with rifampicin (RF) or with rifampicin after encapsulation in phospholipid micelles (nanosome/rifampicin) (NRF) were studied electrochemically. Screen-printed electrodes (SPEs) modified by stable dispersions of multi-wolled carbon nanotubes (MWCNTs) in aqueous solution of poly(1,2-butadiene)-block-poly(2-(dimethylamino)ethyl methacrylate) (PB290-b-PDMAEMA240) diblock copolymer were used for quantitative electrochemical investigation of direct electrochemical oxidation of guanine at E = 0.591 V (vs. Ag/AgCl) and adenine at E = 0.874 V (vs. Ag/AgCl) of dsDNA and its change in the presence of RF or NRF. Due to RF or NRF interaction with dsDNA, the differential pulse voltammetry (DPV) peak currents of guanine and adenine decreased and the peak potentials shifted to more positive values with increasing drug concentration (RF or NRF). Binding constants (Kb) of complexes RF-dsDNA and NRF-dsDNA were calculated based on adenine and guanine oxidation signals. The Kb values for RF-dsDNA were 1.48 × 104 M-1/8.56 × 104 M-1, while for NRF-dsDNA were 2.51 × 104 M-1/1.78 × 103 M-1 (based on adenine or guanine oxidation signals, respectively). The values of Kb revealed intercalation mode of interaction with dsDNA for RF and mixed type of interaction (intercalation and electrostatic mode) for NRF. The estimated values of ΔG (Gibbs free energy) of the complex formation confirmed that drug-dsDNA interactions are spontaneous and favourable reactions.

Chlorin e6 embedded in phospholipid nanoparticles equipped with specific peptides: Interaction with tumor cells with different aminopeptidase N expression.

A promising direction in Biopharmaceuticals is the development of specific peptide-based systems to improve drug delivery. This approach may increase tumor specificity and drug penetration into the target cell. Similar systems have been designed for several antitumor drugs. However, for photodynamic therapy drugs, such studies are not yet enough. Previously, we have developed a method of inclusion of chlorin e6 (Ce6), a photosensitizer used in photodynamic therapy, in phospholipid nanoparticles with a diameter of up to 30 nm, and reported an increase in its effectiveness in the experiments in vivo. In this work, we propose to modify a previously developed delivery system for Ce6 by the addition of cell-penetrating (R7) and/or targeting NGR peptides. The interaction of the compositions developed with HepG2 and MCF-7 tumor cells is shown. The expression of CD13 protein with affinity to NGR on the surface of these cells has been studied using flow cytometry. The expression of this protein on the HepG2 cells and its absence on MCF-7 was demonstrated. After incubation of tumor cells with the resulting Ce6 compositions, we evaluated the cellular accumulation, photoinduced, and dark cytotoxicity of the drugs. After irradiation, the highest level of cytotoxicity was observed when R7 peptide was added to the system, either alone or in combination with NGR. In addition to R7, the NGR-motif peptide increased the internalization of Ce6 in HepG2 cells without affecting its photodynamic activity. In this work we also discuss possible mechanisms of action of the cell-penetrating peptide when attached to phospholipid nanoparticles.